Theobroma cacao L. is a tropical tree of important economic interest for many countries. Cocoa is mainly allogamous and propagated from cross-pollinated seeds leading to plantations which are often established with unselected seedlings resulting in great heterogeneity, whereby only 10% to 20% of the trees produce 60% to 80% of the cocoa beans. Therefore vegetative propagation of selected plants would be desirable, but conventional methods such as cutting or grafting often present growth defaults. In such clonal plantations, cocoa plants are shorter and tend to present an increased growth of side shoots, as well as some branches being very close above the soil. Pruning is needed in order to correct and lift the crown of such trees. Moreover, these propagation methods are difficult to apply on a large-scale commercial basis.
Somatic embryogenesis is a type of vegetative propagation based on plant cell totipotency which offers a powerful alternative to other vegetative propagation methods, e.g. cutting or grafting.
Regardless of the plant species, somatic embryogenesis generally involves    a) induction of embryogenic calli followed by their identification and selection by physical isolation,    b) multiplication of embryogenic cells,    c) regeneration of large numbers of embryos from these cells (embryogenic phase), and    d) conversion of these embryos into mature embryos able to regenerate a plant.
Propagation of Theobroma cacao L. by somatic embryogenesis in solid gel media is known using cocoa flower buds which are subjected to    a) primary embryogenesis in a Petri dish for 9 weeks in the dark at 25° C. in a suitable solid gel culture medium causing induction and expression to produce primary embryos,    b) secondary embryogenesis in a Petri dish    i) for 9-16 weeks in the dark at 25° C. in a suitable solid gel culture medium to produce embryogenic callus followed by    ii) 2×3 weeks (whereby the calli are transplanted and sub-cultivated in a fresh medium after 3 weeks) in the dark at 25° C. in a suitable solid gel culture medium causing expression to produce further new secondary embryos,    c) maturation of the secondary embryos into plantlets for 4-6 weeks in the light at 30° C./25° C. in a solid gel maturation medium,    d) in vitro development for 4-8 weeks in the light at 25° C. in a solid gel culture medium.
The developed plantlets are then transplanted in the greenhouse for plant acclimatization, then the nursery before going to field where they grow into cocoa trees.
The compositions of the basal culture media are well known to persons skilled in the art and they are made solid by the use of a gel such as agar or gelrite. The culture medium may be any one of those described in Driver & Kuniyuki, Hortscience 19 (1984), 507-509; Yasuda, Fuji and Yamaguchi, Plant Cell Physiol. 26 (1985), 595-597; Murashige T. and Skoog., Physiol. Plant. 15 (1962), 473-497, Berthouly M. and Michaux-Ferriere N., Plant Cell Tiss. Org. Cult. 44 (1996), 169-176 and Halperin, W. 146 (1964), 408-410, Lloyd & Mc Cown, WPM, Basal salts Int. Plant Prop. Soc. Proc. Vol. 30 (421-427) 1981 Vitamins Mullin & al. Phytopath. Vol. 64 (1425-1429) 1974, which documents are incorporated herein by way of reference. The composition of the basal media and some growth hormones for the propagation of Theobroma cacao L. by somatic embryogenesis is not the same as for coffee plants but suitable media compositions would be readily ascertainable by persons skilled in the art.
Successful multiplication of the embryonic calli by somatic embryogenesis using a solid gel nutrient medium has permitted a significant production of “in vitro” cocoa trees which are more vigorous (trunk diameter), produce earlier fruiting, are earlier in bearing pods, are more drought tolerant, give an improved yield of the first crops of the trees produced, and only require half the pruning work, compared with cocoa trees derived by grafting or cutting. However, it is still desirable to improve the propagation even further in order to grow cocoa trees on a commercial scale.
Although vegetative propagation of coffee plants by somatic embryogenesis using both solid nutrient culture media and liquid nutrient culture media is known, the use of liquid nutrient culture media for the vegetative propagation of cocoa plants by somatic embryogenesis is not known. Cocoa embryos are larger than coffee embryos and much more fragile and it is important to limit the handling of the embryos which is carried out using forceps and leads to high labour costs. This is different from coffee because coffee embryos produce smaller cotyledons and thus can be transferred from one medium to another one without damaging them.